Mouse stromal cell lines (MSS lines) have been established from the spleens of newborn mice in culture at a low serum concentration. These MSS lines support the proliferation and differentiation of the erythroid progenitor cells from mouse fetal livers and bone marrow in a semisolid medium in the presence of erythropoietin. Larger colonies of over 1,000 benzidine-positive erythroid cells were developed from the fetal liver cells on the MSS cell layers after 6 days of incubation. These layers also support the maturation of the erythroid cells since the enucleation process of the latter was observed in large erythroid colonies. Metabolically active MSS cells are apparently required to support the proliferation and differentiation of the erythroid progenitor cells, because neither the MSS cells inactivated with fixation nor the conditioned media of MSS cells promoted the erythroid colony formation. These studies demonstrate that MSS lines specifically support the proliferation and differentiation of the erythroid progenitor cells in vitro and that stroma cells may have a critical function in blood formation in the mouse spleen.